The present invention relates generally to the treatment of neurons following NS injury that may result from surgery, trauma, compression, contusion, transection or other physical injury, from vascular pharmacologic or other insults including hemorrhagic or ischemic damage or from neurodegenerative or other neurological diseases. More specifically, the invention relates to the preparation and use of devices for transferring neuronal therapeutic agents and/or DNA encoding neuronal therapeutic agents into the NS, including devices that are gene activated matrices, to alter the function, gene expression or viability of neuronal cells therapeutically. The invention further relates to administration of such devices, including administration of matrices containing useful genes.
Neuronal regeneration and restoration of neural connectivity within denervated tissues may be desirable events following acute or chronic nervous system (NS) injury resulting from physical transection/trauma, contusion/compression or surgical lesion, vascular pharmacologic insults including hemorrhagic or ischernic damage, or from neurodegenerative or other neurological diseases. Promotion of NS neuronal protection, neuronal survival and axon generation are well controlled processes that mainly originate during embryonic development and may persist through adulthood.
The stability of neuronal networks depends in part on the availability of a variety of specific architectural and biochemical cues in the neuronal environment that maintain neuronal projections, including axons. In the adult NS, the viability of neurons is maintained by the continuous retrograde flow of neurotrophic factors from the distal neuronal target to the neuronal cell body (perikaryon). Interruption of neural connections by physical severance of axons disconnects neuron from target and threatens neuronal survival.
Because of the spatiotemporal regulation of cues, including neurotrophic factors, essential for the maintenance of neural networks, axonal regrowth following NS injury is impaired by the absence of one or more appropriate stimuli in the vicinity of the damaged neuron. For example, neurotrophins (NT, discussed below) may be primary determinants of neuronal regeneration, and neurotrophin availability can be a primary limiting factor for axonal regrowth. Damaged neurons may initially start to regenerate axons, as a response to transient and regulated increases in the expression of neurotrophic factors, but regrowth is usually aborted within 14 days as intracellular stores of neurotrophin in the perikaryon are exhausted. Regrowth may also be inhibited in part by the deposition of fibrotic scar tissue during the course of wound healing. The synthesis and release of growth factors by mesenchymal and glial cells within the fibrotic scar may create localized microenvironments, or xe2x80x9csinksxe2x80x9d, having high growth factor concentrations. Because neurotrophin dependent axonal regeneration obligatory proceeds up a concentration gradient of the neurotropic factor, axonal entrapment within a growth factor sink may result. Following axonal injury, a neuron may be deprived of essential maintenance signals (e.g., neurotrophic factors that ordinarily would be supplied from distal target regions through an intact axon), and may die. Consequently, reconnection of neural pathways is prevented and functional recovery may be compromised.
Efforts to induce axonal regrowth following NS injury have included direct or indirect administration of neurotrophic compounds at or near lesion sites. According to such approaches, a neurotrophic compound may be directly applied at or near a lesion, or may be indirectly introduced to the damaged tissue by a transplanted cell secreting the neurotrophin(s). These methods often produce localized sinks of high neurotrophin concentration at the lesion site in which axons may become entrapped. Thus, axonal extension beyond the lesion and along the damaged projection tracts may be impossible. Failure to re-establish neural connections and the ensuring neuronal atrophy may result in complete loss of function.
Another approach designed to promote axonal regrowth after NS injury utilizes recombinant viral vectors to deliver therapeutic genes encoding neurotrophic factors. Depending on the viral vector construct and delivery vehicle used, such approaches may under certain circumstances, (i) elicit inappropriate antiviral immune responses, (ii) promote undesirable viral toxic effects, (iii) have limited efficacy due, for example, to inefficiency of genetically altered viral gene promoter sequences, (iv) be tumorigenic and/or (v) lack specificity regarding the cell type to which therapeutic genes are delivered. Poor targeting of such recombinant viral vectors to specific cell types, for example, may limit the value of such an approach and may establish localized accumulations of therapeutic gene products at the site of vector delivery, giving rise to the problems associated with localized growth factor sinks and axonal entrapment.
In view of these and other problems associated with neuroregenerative therapy, there is a compelling need for improved and more effective treatments that are free of the above disadvantages.
The present invention exploits the use of gene activated matrices that, when administered into a NS lesion site or along the axonal projection tract proximal to a lesion, deliver high amounts of nucleic acids encoding a desired neuronal therapeutic product by retrograde axonal transport to distant, targeted neuronal cell perikaryons without inducing localized sinks of active product that may lead to axonal entrapment, while providing other related advantages.
The compositions and methods of the present invention may be useful wherever neuronal regeneration and restoration of connectivity within neural networks is sought, for example following any acute or chronic NS injury resulting from physical transection/trauma, contusion/compression or surgical lesion, vascular pharmacologic insults including hemorrhagic or ischemic damage, or from neurodegenerative or other neurological diseases.
NS injury resulting from physical transection/trauma, vascular pharmacologic insults and/or neurological diseases may further include mechanical insult and may also include NS injury resulting from burns or other chemical exposure. Such exposure may include but need not be limited to exposure to toxic compounds such as carbon monoxide or other metabolic poisons, or exposure to free radicals, as may also accompany aging or contribute to the pathogenesis of neurodegenerative disease. For example, increased levels of reactive oxygen species may be present, and may correlate with sites of neurodegeneration, in diseases such as Alzheimer""s disease, Parkinson""s disease or Huntington""s disease.
Interruption of neural connections may be a consequence of acute or chronic NS injury leading to physical severance of axons that threatens neuronal survival, as described above. Accordingly, the compositions and methods of the present invention may delay cell degeneration and cell death by restoring the continuous retrograde flow of neurotrophic factors, from distal neuronal targets to neuronal perikarya, that is essential for maintenance of neural networks.
A considerable amount of work has been directed to the development of biocompatible matrices for use in medical implants, including those specifically for connective tissue implantation such as in bone or wound healing. In context of the present invention, a matrix may be employed in association with the gene or DNA coding region encoding a neuronal therapeutic agent in order to easily deliver the gene to the site of NS injury. The matrix is thus a xe2x80x9cbiofillerxe2x80x9d that provides a structure for the regulated regeneration of neuronal axons. Such matrices may be formed from a variety of materials presently in use for implanted medical applications.
According to the present invention, compositions and methods are provided for matrix mediated delivery of agents, and in preferred embodiments neuronal therapeutic encoding agents, that promote neuronal regeneration and survival.
In one aspect the invention provides a device for promoting neuronal regeneration, comprising a gene activated matrix comprising a biocompatible matrix and at least one neuronal therapeutic encoding agent having an operably linked promoter. In another aspect the invention provides a device for promoting neuronal survival, comprising a gene activated matrix comprising a biocompatible matrix and at least one neuronal therapeutic encoding agent having an operably linked promoter. In certain embodiments of these aspects, the promoter is an inducible promoter and in certain embodiments the promoter is a tissue specific promoter. In certain embodiments the promoter is GAP43 promoter, GFAP promoter, neuron specific enolase promoter, FGF-receptor promoter, elastase I gene control region, immunoglobulin gene control region, alpha-1-antitrypsin gene control region, beta-globin gene control region, myelin basic protein gene control region, myosin light chain 2 gene control region, RSV promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV promoter, MLP adenovirus promoter, MMTV LTR promoter, CMV promoter, metallothionein promoter, a promoter having at least one cAMP response element, tie promoter, VCAM-1 promoter, alpha V-beta 3 integrin promoters, ICAM-3 promoter, CD44 promoter, CD40 promoter, notch 4 promoter, or an event type-specific promoter. In other embodiments the promoter is a neuronal cell specific promoter, which in certain further embodiments may be GAP43 promoter, FGF receptor promoter or neuron specific enolase promoter.
In certain embodiments, the neuronal therapeutic encoding agent encodes a neurotrophic factor, which in certain further embodiments may be a member of the neurotrophin family and in certain other further embodiments may be a member of the FGF family. In certain of these embodiments the neurotrophic factor may be nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), cardiotrophin-1 (CT-1), choline acetyltransferase development factor (CDF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM); fibroblast growth factor-1 (FGF-1), FGF-2, FGF-5, glial cell-line-derived neurotrophic factor (GDNF), insulin, insulin-like growth factor-1 (IGF-1), IGF-2, interleukin-6 (IL-6), leukemia inhibitor factor (LIF), neurite promoting factor (NPF), neurotrophin-3 (NT-3), NT4, platelet-derived growth factor (PDGF), protease nexin-1 (PN-1), S-100, transforming growth factor-xcex2 (TGF-xcex2), or vasoactive intestinal peptide (VIP).
In some embodiments, the neuronal therapeutic encoding agent encodes an inhibitor of an antagonist of axonal generation or regeneration, and in certain further embodiments the inhibitor of an antagonist of axonal generation or regeneration is an inhibitor of TGF-beta. In certain embodiments, the inhibitor of TGF-beta is decorin, a TGF-beta inhibitory chemokine, an anti-TGF-beta antibody, an antisense TGF-beta oligonucleotide, a TGF-beta gene specific ribozyme or a mutated TGF-beta. In certain embodiments, the TGF-beta inhibitory chemokine is an ELR containing member of the CXC chemokine family. In certain embodiments, the ELR containing member of the CXC chemokine family is selected from the group consisting of interleukin-8, ENA-78, GROxcex1, GROxcex2 and GROxcex3. In certain embodiments, the inhibitor of TGF-beta is decorin. In certain embodiments, the inhibitor of TGF-beta is an anti-TGF-beta antibody. In certain embodiments, the inhibitor of TGF-beta is a mutated TGF-beta.
In some embodiments, the neuronal therapeutic encoding agent is non-covalently associated with the gene activated matrix. In certain embodiments, the neuronal therapeutic encoding agent is adsorbed to the gene activated matrix, and in certain other embodiments the neuronal therapeutic encoding agent is absorbed in the gene activated matrix. In certain embodiments, the neuronal therapeutic encoding agent is capable of inducing neuronal axonal generation or regeneration.
It is another aspect of the invention to provide a device for promoting neuronal regeneration, comprising a gene activated matrix, at least one support cell, and at least one neuronal therapeutic encoding agent having an operably linked promoter. It is yet another aspect of the invention to provide a device for promoting neuronal survival, comprising a gene activated matrix, at least one support cell, and at least one neuronal therapeutic encoding agent having an operably linked promoter. In certain embodiments of either of these aspects the support cell is a Schwann cell, and in certain other embodiments the support cell is an oligodendrocyte. In certain embodiments the support cell is an astrocyte and in certain embodiments the support cell is a microglial cell. In certain embodiments the support cell is a fibroblast. In certain embodiments the support cell is a macrophage. In certain embodiments the support cell is an inflammatory cell which may be a macrophage, a neutrophil, a monocyte, a granulocyte or a lymphocyte.
In certain embodiments of the invention, the neuronal therapeutic encoding agent is capable of maintaining axonal generation or regeneration. In certain embodiments the gene activated matrix is an implant for a neuronal injury site. In certain embodiments the gene activated matrix is formed upon administration. In certain embodiments the gene activated matrix is administered to a neuronal injury site. In certain embodiments the gene activated matrix is a composition selected that is a solution, a paste, a suspension, a powder, a semisolid, an emulsion or a gel. In certain preferred embodiments, the gene activated matrix is a paste. In certain embodiments the neuronal therapeutic encoding agent is a nucleic acid molecule, a vector, an antisense nucleic acid molecule or a ribozyme.
In some embodiments of the invention, the device further comprises a targeting agent, which is complexed with the neuronal therapeutic encoding agent and is capable of binding a neuronal cell surface receptor. In certain other embodiments, the targeting agent is conjugated to the neuronal therapeutic encoding agent and is capable of binding a neuronal cell surface receptor. In certain other embodiments, the targeting agent is complexed with the neuronal therapeutic encoding agent and is capable of binding a repair cell surface receptor. In certain other embodiments, the targeting agent is conjugated to the neuronal therapeutic encoding agent and is capable of binding a repair cell surface receptor. In certain other embodiments, the targeting agent is complexed with the neuronal therapeutic encoding agent and is capable of binding extracellular matrix. In certain other embodiments, the targeting agent is conjugated to the neuronal therapeutic encoding agent and is capable of binding extracellular matrix. In certain other embodiments, the device further comprises a nucleic acid binding domain, wherein the nucleic acid binding domain binds to a nucleic acid sequence that forms a portion of the neuronal therapeutic encoding agent. In certain other embodiments, the device further comprises at least one linker that may be a cleavable linker, a linker that provides an intracellular protein sorting peptide sequence, a linker that reduces steric hindrance, a linker that provides a nuclear translocation signal or a linker that possesses a nucleic acid condensing ability. In certain other embodiments, the device contains sub-physiologic amounts of a neuronal therapeutic agent. In certain other embodiments, the device contains physiologic amounts of a neuronal therapeutic agent.
In certain other embodiments of the above described aspects of the invention, the device further comprises a conduit having a lumen. In certain embodiments, the conduit comprises the gene activated matrix and in certain other embodiments, the lumen contains the gene activated matrix. In certain embodiments, the conduit comprises a bioabsorbable material, which in certain further embodiments may be a material comprising gene activated matrix, type I collagen, laminin, polyglycolic acid, glycolide trimethylene carbonate (GTMC), poly (L-lactide-co-6-caprolactone), glycoproteins, proteoglycans, heparan sulfate proteoglycan, nidogen, glycosaminoglycans, fibronectin, epidermal growth factor, fibroblast growth factor, nerve growth factor, cytokines, or DNA encoding growth factors and cytokines.
In certain other embodiments, the conduit comprises a non-bioabsorbable material, which in certain further embodiments is be polyamide, polyimide, polyurethane, segmented polyurethane, polycarbonate or silicone. In certain other embodiments, the non-bioabsorbable material comprises an etched microporous synthetic polymer surface. In certain embodiments the conduit is tubular.
Turning to another aspect of the invention, a method is provided for transferring a neuronal therapeutic encoding agent into a neuronal cell, comprising contacting a neuronal cell with any one of the devices just described to effectively transfer the neuronal therapeutic encoding agent into the neuronal cell. In one embodiment, transfer of the neuronal therapeutic encoding agent comprises retrograde axonal transport of the neuronal therapeutic encoding agent. In another embodiment the method further comprises expression of the neuronal therapeutic encoding agent at a neuronal cellular site distinct from a site of contact between the device and the neuronal cell. In another embodiment, the device is contacted with a neuronal cell at a neuronal injury site. In another embodiment, the device is contacted with a neuronal cell in a manner such that axonal generation or regeneration occurs. In a further embodiment, axonal regeneration occurs without axonal entrapment. In another embodiment, the device is contacted with a neuronal cell in a manner that promotes neuronal survival. In a further embodiment, neuronal survival is promoted without axonal entrapment. In certain further embodiments a neural connection is established or reestablished.
It is yet another aspect of the invention to provide a method for transferring a neuronal therapeutic encoding agent into a repair cell, comprising contacting a repair cell with any one of the devices described above to effectively transfer the neuronal therapeutic encoding agent into the repair cell. In one embodiment, the device is contacted with a repair cell at a neuronal injury site, and in another embodiment the device is contacted with a repair cell in a manner such that axonal generation or regeneration occurs. In certain further embodiments axonal generation or regeneration occurs without axonal entrapment. In another embodiment, the device is contacted with a repair cell in a manner that promotes neuronal survival. In a further embodiment, neuronal survival is promoted without axonal entrapment. In certain other embodiments a neural connection is established or reestablished.
In certain embodiments of the method the device contains sub-physiologic amounts of a neuronal therapeutic agent, and in certain other embodiments the device contains physiologic amounts of a neuronal therapeutic agent.
In still another aspect, the invention provides a method of preparing a gene activated matrix for promoting neuronal regeneration and survival, comprising contacting a neuronal therapeutic encoding agent with a biocompatible matrix such that the neuronal therapeutic encoding agent associates non-covalently with the matrix. In one embodiment, the neuronal therapeutic encoding agent is adsorbed to the gene activated matrix, and in another embodiment the neuronal therapeutic encoding agent is absorbed in the gene activated matrix. In certain embodiments the neuronal therapeutic encoding agent is a nucleic acid molecule, a vector, an antisense molecule or a ribozyme.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entireties.